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Self-assembled nucleic acid nanotube preparation as well as preparation method and application thereof

A technology of nucleic acid nanotubes and nano-preparations, which is applied in drug combinations, pharmaceutical formulations, cardiovascular diseases, etc., to achieve the effects of reducing mRNA and protein expression levels, inhibiting abnormal proliferation, and enhancing autophagy

Inactive Publication Date: 2015-04-29
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, how to design and develop a clinically applicable siRNA delivery system for the treatment of pulmonary arterial hypertension is still a huge challenge.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Synthesis of tubular nucleic acid nanoformulations

[0055] Dry powder of each sequence of mTOR siRNA (SEQ ID NO: 1), L (SEQ ID NO: 2), M (SEQ ID NO: 3), S (SEQ ID NO: 4) and S'(SEQ ID NO: 5) The samples were diluted to 0.2μg / μl with sterile and enzyme-inactivated deionized water; the L, M, S, S′ solutions were added to TAE-Mg 2+ Compounded in buffer (pH 8.0), nucleic acid nanotubes (DNA nanotubes) are self-assembled and synthesized under the following conditions: 95℃ for 5 min, 65℃ for 30 min, 50℃ for 30 min, 37℃ for 30 min, then 22℃ for 30 min . Subsequently, the determined dose of mTOR siRNA was added into the nanotube solution and left for another 30 minutes. Obtain 500 μl of tubular nanotube preparation solution. The molar ratio of mTOR siRNA, L, M, S, S'in the preparation is 6:1:3:3:3, and can be used as a medicine for treating pulmonary hypertension.

[0056] The atomic force microscope scanning image of the tubular nucleic acid nano preparation structur...

Embodiment 2

[0059] Example 2: Effects of different doses of transfection reagents on the transfection of tubular nucleic acid nanoformulations

[0060] The PASMC in the logarithmic growth phase was digested with 0.25% trypsin, the cells climbed into the slide, and the cells were seeded in a 24-well plate (built-in cover glass), placed at 37°C, 5% CO 2 After 48 hours of incubation in the incubator, it can be used for transfection when the cell density reaches 50%-60%; the experiment group is: tubular nucleic acid nano preparation (50nM): X-tremeGENE siRNA ratio is 1: 0, 1: 0.25, 1:0.5, 1:1 and 1:2, the DNA nanotube nanoformulations carrying mTOR siRNA were transfected according to the instructions of X-tremeGENE siRNA transfection reagent; after transfection of the nanoformulations, placed at 37℃, 5% CO 2 Incubate in an incubator for 24 hours. After that, discard the 24-well plate medium, rinse gently with 37℃PBS for 3min×3 times; add 1ml 4% paraformaldehyde along the culture plate wall, fix f...

Embodiment 3

[0063] Example 3: Effect of different doses of tubular nucleic acid nano preparations on cell transfection

[0064] The concentration of design tubular nucleic acid nano preparations is divided into five concentrations: 0nM, 12.5nM, 25nM, 50nM and 100nM. During laser confocal observation, a single Cy3-mTOR siRN concentration of 12.5nM is set as the control group. In flow cytometry detection For each concentration, a single Cy3-mTOR siRNA was used as a control, and a blank control group was set. The laser confocal observation experiment of cell transfection efficiency and the experiment operation of the flow cytometer to detect the average fluorescence density are the same as those in Example 2.

[0065] As shown in Fig. 5A and Fig. 5B by laser confocal, Cy3-mTOR siRNA carried by tubular nucleic acid nanocarriers showed red fluorescence distribution in cells, and with the increase of tubular nucleic acid self-assembled nano preparations, the amount of DNA nanotubes entering cells in...

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Abstract

The invention relates to a self-assembled nucleic acid nanotube preparation as well as a preparation method and application thereof. The preparation comprises mTOR siRNA as shown in a sequence SEQ ID NO: 1, L as shown in sequence SEQ ID NO: 2, M as shown in sequence SEQ ID NO: 3, S as shown in sequence SEQ ID NO: 4, and S' as shown in sequence SEQ ID NO: 5; nucleic acid in which single-strand nucleic acid molecular bases supplement each other and are paired is crosslinked into a tubular nucleic acid nanotube preparation. The preparation enables double coiling of siRNA and is self-assembled into a nanotube system, so that siRNA is difficultly degraded by nuclease, and a compound nanotube structure can be generated; therefore, the advantage that nanoparticles are easily absorbed by cells is brought into full play, and the transfection efficiency of cells can be obviously improved; the siRNA can stably enter the cell structure, the original biological activity can be remained, the autophagy and cell proliferation can be adjusted and controlled, the abnormal growth of pulmonary arterial smooth muscle cells can be effectively inhibited, and the self-assembled nucleic acid nanotube preparation can be applied to preparation of medicines for treating pulmonary hypertension.

Description

Technical field [0001] The present invention relates to a biological preparation, in particular to a tubular nucleic acid nano preparation containing siRNA, and a preparation method and application. Background technique [0002] The development of universal drug carriers is the most important research topic of nanomedicine. Previous studies have reported many drug delivery systems, such as synthetic high molecular polymers, cationic liposomes, carbon nanotubes, cyclodextrin materials, nanoparticles, viral capsids, etc. The effectiveness of these delivery systems has been clearly demonstrated, however, most of them are unnatural or exogenous molecules, lack tissue specificity and have potential toxicity. Recent research on the structure of DNA makes it possible to be further used in the field of biology. DNA is a natural component in the human body. It is relatively stable and can be biodegraded without producing immunogenicity. Therefore, self-assembled DNA nanostructures have...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/14A61K48/00A61K31/7088A61P11/00A61P9/12
Inventor 王关嵩尤再春王应明钱航杨俊俊
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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